Abstract
X-linked inhibitor of apoptosis protein (XIAP) suppresses apoptosis and plays key roles in the development, growth, migration, and invasion of cancer cells. Therefore, XIAP has recently attracted much attention as a potential antineoplastic therapeutic target, requiring elucidation of the molecular mechanisms underlying its biological activities. Here, using shRNA-mediated gene silencing, immunoblotting, quantitative RT-PCR, anchorage-independent growth assay, and invasive assay, we found that XIAP's RING domain, but not its BIR domain, is crucial for XIAP-mediated up-regulation of c-Myc protein expression in human bladder cancer (BC) cells. Mechanistically, we observed that the RING domain stabilizes c-Myc by inhibiting its phosphorylation at Thr-58 and that this inhibition is due to activated ERK1/2-mediated phosphorylation of glycogen synthase kinase-3β (GSK-3β) at Ser-9. Functional studies further revealed that c-Myc protein promotes anchorage-independent growth and invasion stimulated by the XIAP RING domain in human BC cells. Collectively, the findings in our study uncover that the RING domain of XIAP supports c-Myc protein stability, providing insight into the molecular mechanism and role of c-Myc overexpression in cancer progression. Our observations support the notion of targeting XIAP's RING domain and c-Myc in cancer therapy.
Highlights
X-linked inhibitor of apoptosis protein (XIAP) suppresses apoptosis and plays key roles in the development, growth, migration, and invasion of cancer cells
We found that the XIAP RING domain could stabilize c-Myc protein through inhibition of its phosphorylation at Thr-58, which is mediated by increased phosphorylation of glycogen synthase kinase-3 (GSK-3) at Ser-9 due to ERK1/2 activation
The results obtained from comparison of c-Myc expression between ⌬RING mouse embryonic fibroblasts (MEFs) and WT MEFs indicated that c-Myc protein expression in XIAP RING domain– deficient MEFs(⌬RING) was impaired as compared with that in WT-MEFs (Fig. 1G)
Summary
Using shRNA-mediated gene silencing, immunoblotting, quantitative RT-PCR, anchorage-independent growth assay, and invasive assay, we found that XIAP’s RING domain, but not its BIR domain, is crucial for XIAP-mediated up-regulation of c-Myc protein expression in human bladder cancer (BC) cells. Functional studies further revealed that c-Myc protein promotes anchorage-independent growth and invasion stimulated by the XIAP RING domain in human BC cells. The findings in our study uncover that the RING domain of XIAP supports c-Myc protein stability, providing insight into the molecular mechanism and role of c-Myc overexpression in cancer progression. We found that the XIAP RING domain could stabilize c-Myc protein through inhibition of its phosphorylation at Thr-58, which is mediated by increased phosphorylation of glycogen synthase kinase-3 (GSK-3) at Ser-9 due to ERK1/2 activation. C-Myc is an effector for promotion of anchorage-independent growth and invasion by XIAP RING domain in BC cells
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